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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 3  |  Issue : 2  |  Page : 60-64

Liver function tests as a measure of hepatotoxicity in areca nut chewers


Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Institute, Bengaluru, Karnataka, India

Date of Web Publication16-Jun-2016

Correspondence Address:
Kanika Singroha
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Institute, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2348-2915.184210

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  Abstract 

Background: Areca nut has been listed as a carcinogenic agent in humans and is linked to cancers of oral cavity, gastrointestinal tract, and hepatobiliary system. Liver function tests (LFTs), the estimation of enzymes specific to the hepatic system, give an assessment of its cellular integrity, and functionality. Aim and Objectives: This study aimed to evaluate the state of the liver in patients consuming areca nut and its products over a period. Materials and Methods: LFTs were carried out on 10 nonareca nut chewers and thirty patients with a history of areca nut, quid or a combination of tobacco and areca nut chewing, extending from 6 months to 30 years. The LFTs included estimation of aspartate transaminase (AST), alanine transaminase, alkaline phosphatase (ALP), direct bilirubin, albumin, and total protein content. A comparative analysis was done for each biochemical marker with duration, form (betel nut alone, quid, and betel nut with tobacco), and frequency of chewing areca nut. Results: A mild increment in AST was seen in 33.3% cases. Statistically significant association (P < 0.05) was observed between the control and cases for AST, ALP, and total protein content. A significant positive Pearson's correlation (+0.417) was obtained for a form of areca nut chewing (areca nut and tobacco) and AST. A significant negative Pearson's correlation (−0.05) was observed between total protein content and form of chewing (areca nut and tobacco). Conclusion: The results of the study seem to indicate that even long-term chewing of areca nut is not hepatotoxic. Minor alterations in LFTs were well within limits.

Keywords: Areca nut, hepatotoxicity, liver function tests


How to cite this article:
Singroha K, Kamath VV. Liver function tests as a measure of hepatotoxicity in areca nut chewers. J Dent Res Rev 2016;3:60-4

How to cite this URL:
Singroha K, Kamath VV. Liver function tests as a measure of hepatotoxicity in areca nut chewers. J Dent Res Rev [serial online] 2016 [cited 2022 Nov 27];3:60-4. Available from: https://www.jdrr.org/text.asp?2016/3/2/60/184210


  Introduction Top


Alcohol consumption and the habit of chewing betel quid, containing fresh, dried or cured areca nut, catechu, slaked lime, and flavoring ingredients wrapped in betel leaf is a common component of cultural fabric in the geographical areas of South-East Asia including the Indian subcontinent.[1],[2] India has the largest area under areca nut cultivation.[3] It is consumed either as betel nut per se or as a quid that comprises areca nut wrapped in betel leaf. It is also consumed along with tobacco.[4] Areca nut has been listed as a carcinogenic agent in humans and is linked to cancers of oral cavity, gastrointestinal tract, and hepatobiliary system.[5] Chewing betel quid leads to cyanoethylation and methylation of liver DNA and produces nitrosamines that cause liver carcinoma due to genotoxicity of hepatocytes.[6]

Liver disease is a worldwide problem. The liver is an organ of paramount importance as it plays an essential role in maintaining the biological equilibrium of vertebrates. The spectrum of function includes: Regulation of xenobiotics to which the organ is exposed directly or indirectly, metabolism of lipids, carbohydrates and protein, and blood coagulation. Liver function tests (LFTs), the estimation of enzymes specific to the hepatic system, give an assessment of its cellular integrity, and functionality. The LFTs included the estimation of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), direct bilirubin, albumin, globulin, and total protein estimation.[7] The common LFTs and their significance have been enlisted in [Table 1]. Descriptive analysis of the liver biochemical markers in the Group I of 30 areca nut chewers and 10 healthy individuals in Group II regarding frequency, duration, and form of areca nut chewed was done. Comparative analysis was done for each LFT between Group I and Group II through Mann–Whitney U-test.
Table 1: Key biochemical markers in hepatobiliary system and functions

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This study evaluated the hepatotoxic effects of areca nut and its various combinations in persons habituated to the same. Correlations of the LFTs with duration, type, and frequency of habit were also done. The study was carried out on an open-ended hypothesis that the effects of areca nut may contribute to changes in liver health and status as judged by LFTs.


  Materials and Methods Top


The study included total of n = 40 subjects with n = 30 patients with a history of areca nut, quid or a combination of tobacco, and areca nut chewing, extending from 6 months to 30 years in Group I as study group and n = 10 as healthy individuals in Group II as control group and the subjects included were outpatients in Dr. Shyamala Reddy Dental College and Research Centre. Informed consent was obtained from the subjects in both groups. Detailed case history was recorded for all the subjects. The subjects fulfilling the inclusion and exclusion criteria were subjected to collection of a blood sample for LFT estimation. Ethical clearance was applied for, and the study was cleared by the Ethical Committee of the Institution. The age-wise distribution of the subjects was done [Table 2]. There were n = 22 males and n = 8 females in the study group [Table 3]. The frequency of contained subjects ranging from subjects consuming twice a day to 8 times a day [Table 4]. Form in which betel nut is chewed, and duration of chewing was recorded [Figure 1] and [Figure 2]. Strict inclusion criteria were that no individual, in both the groups, should have had a history of any form of liver ailment in the past 10 years. Only teetotallers were included in both the study groups. Information regarding alcohol consumption, medical history, were collected so as ensure that no superimposed liver ailment existed in any of the subjects. A Mann–Whitney U-test was conducted to observe the difference in the LFT levels among the two groups at P < 0.05. The LFTs included estimation of AST, ALT, ALP, direct bilirubin, albumin, globulin, and total protein estimation. Comparative analysis was done for each biochemical marker with duration, form, and frequency of chewing areca nut. The blood samples were collected by laboratory technician following infection protocol. A volume of 5 ml whole blood from the subject was collected in a vacutainer containing a clot accelerator. The blood sample was centrifuged for 10 min, and serum was separated. Serum was analyzed for a panel of LFT consisting of an evaluation of various enzymes and proteinsin vitro through diagnostic kits manufactured by Euro Diagnostics. Euro Diagnostics semi auto analyzer was used for the assessment of the samples. The panel of tests with their normal reference range as mentioned in the diagnostic kit [Table 5].
Table 2: Age-wise distribution of the study Groups I and II

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Table 3: Gender-wise distribution of the subjects

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Table 4: Frequency of consumption various forms of areca nut

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Figure 1: Distribution of cases based on form of areca nut consumption (comprising areca nut consumers, areca nut and tobacco, quid (arecanut with betel leaf)

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Figure 2: Distribution of study participants based on duration of arecanut chewing

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Table 5: The panel of tests with their normal reference range as mentioned in the Euro-Diagnostic diagnostic kit

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  Results Top


A total of n = 40 blood samples were collected where n = 30 in cases (areca nut consumers) as the study group in Group I and n = 10 (healthy individuals) were in control group as Group II. The study included 22 male and 8 female subjects in the cases. The frequency of consumption ranged from twice a day to 8 times a day, with most of the subjects consuming n = 9 consuming 6 times a day. The subjects consumed a combination of areca nut along with tobacco n = 12, followed by areca nut alone n = 9, and quid n = 9 [Figure 1], and areca nut for more than 15 years n = 7 [Figure 2].

Panel of LFT showed varying results for each enzyme. A mild increment in AST was seen in 33.3% cases. Statistically significant association (P = 0.031) was observed between the control (mean = 24.20) and cases (mean = 33.80) for AST [Table 6]. Statistically significant association (P = 0.02, P < 0.05) was observed for ALP too between control (mean = 108) and cases (mean = 155.38) [Table 7]. The levels of ALT remained unaltered. Statistically significant association was observed for total protein count as well (P = 0.001) with control having (mean = 7.52) and cases (mean = 6.99) [Table 8].
Table 6: Correlations statistics between aspartate transaminase, frequency, duration, and form of the arecanut products use

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Table 7: Correlations statistics between alkaline phosphatase, frequency, duration, and form of the arecanut products use

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Table 8: Correlations statistics between total protein, frequency, duration and form of the arecanut products use

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A significant positive Pearson's correlation (+0.417) was obtained for a form of areca nut chewing (areca nut and tobacco) and AST. It indicates that AST levels increased with this combination of chewing. A significant negative Pearson's correlation (−0.05) was observed between total protein content and form of chewing (areca nut and tobacco) [Table 9].
Table 9: Comparison of parameters between 2 study groups using Mann-Whitney U-test

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  Discussion Top


The consumption of betel nut for its various euphoric effects dates to the dawn of human civilization. India is the second largest producer of areca nut producing 330,000 million tonnes of areca nut per year.[1],[2] A consensus workshop held, in 1996, recommended that the term “quid” should be defined as “a substance, or mixture of substances, placed in the mouth, usually containing at least one of the two basic ingredients, tobacco or areca nut, in raw or any manufactured or processed form.”[8]

Hepatocellular carcinoma (HCC) is the third most common cause of mortality from cancer and the sixth most common cause of cancer incidence worldwide.[9] The structural integrity and functionality of liver can be assessed by LFTs. Assessment of LFTs serves as one of the primary and noninvasive diagnostic help in evaluating the liver health. The common alterations in enzyme levels can be divided into two major subgroups: Hepatocellular predominant and cholestatic predominant. An increase in serum concentrations of aminotransferases is an indication of liver injury, whether acute or chronic. Although both aminotransferases are highly concentrated in the liver, heart, skeletal muscle, kidneys, brain, and red blood cells, have AST diffusely present whereas ALT has low concentrations in skeletal muscle and kidney. Hence, an increase in ALT serum levels is more specific for hepatocellular injury. Decreased albumin levels indicate cirrhosis, thereby acting as a prognostic indicator.[7]

Areca nut has been classified as a human carcinogen (Group I) associated mainly with oral, pharyngeal, esophageal, and liver cancer.[5] The practice of swallowing the saliva containing the toxins of betel nut and other components of the quid contact the upper digestive tract directly. This absorbtion of the areca nut solution increases the effect at target organs other than oral cavity.[10] In this study, mild increase in AST, ALP, and a decrease in total protein content was seen, particularly in the groups chewing a mixture of areca nut along with tobacco and other ingredients, indicating that hepatic damage may be caused in the presence of other ingredients. In the absence of any rise in ALP levels, the status of hepatic injury being related to areca nut composition remains inconclusive. The positive correlation obtained for form of areca nut chewed and rise in LFT levels cannot be overlooked. It has been demonstrated that powdered areca nut placed in the oral cavity of human volunteers gives rise to a rapid appearance of arecoline, the major alkaloid in areca nuts, in blood plasma, indicating systemic absorption of this alkaloid.[11] That endogenous metabolites of areca nut such as nitrosamines and aflatoxin contaminated quids could partly damage liver has been found inin vivo and in vitro studies.[12] Habitual betel nut chewing increases the risk of toxic hepatitis due to the formation of reactive oxygen adducts.[13],[14] In this study, a significant positive Pearson's correlation (+0.417) was obtained for form of areca nut chewing (areca nut and tobacco) and AST. It indicates that AST levels increased with this combination of chewing. A significant negative Pearson's correlation (−0.05) was observed between total protein content and form of chewing (areca nut and tobacco) [Table 6]. It indicates that this combination reduces the protein production efficiency of liver. Review of literature also suggests that immunological suppression of body due to hepatitis B virus/hepatitis C virus (HCV) infections increases the risk of hepatotoxicity.[15]

Although areca nut has been listed as a potent carcinogen in the International Agency for Research on Cancer (IARC) monograph, its beneficial effects too have been documented. Areca husk exhibits antifungal properties through substrate deprivation and membrane disruption of the fungal organism. Tannins form complex with iron ions that interfere with Fenton reaction and exhibit their antioxidant activity.[16] Prevention of inflammation and hepatotoxic disorders induced by free radicals has been reported through procyanidins (major condensed tannins) in areca nut. In this study, the subjects consuming betel nut without tobacco did not show any significant rise in ALT levels though most of them had been chewing for more than 10 years. ALT is more a reliable biochemical marker for accessing the structural integrity of liver than AST as it is contributed by liver alone. Furthermore, no significant rise in the LFT levels of the groups, comprising betel nut chewers alone and the one with betel nut and leaf, was obtained. This may be attributed to the antioxidant activity of the tannins present in the betel nut along with the regenerative capacity of the liver. Studies in the past too indicate the promising anti-carcinogenic role of betel leaf extracts in tobacco-induced cancer in rats.[17] Thein vivo micronucleus test too showed antimutagenic effect of arecanut leaf standard against mutagens such as benzo[a] pyrene and dimethylbenz[a] anthracene.[18]

Surprisingly, in this study, few individuals who had been chewing areca nut for a very long period of 15–20 years with a higher frequency along with the leaf did not show any rise in the LFT levels. (IARC), studies support the hypothesis that areca nut may not be a causative agent for hepatotoxicity per se, rather acts as an associative agent that in the presence of a preexisting condition such as hepatitis increases the susceptibility of the liver to hepatocellular damage. The interaction between HCV and habitual betel quid chewing was assessed in two studies conducted in Taiwan.[19],[20] Sun et al., in 2003, suggested a greater than additive increased the risk of HCC related to the combined effect of HCV infection and betel quid chewing than of either factor alone with a synergy index of 4.2.[19] Tsai et al., in 2001, mentioned a weaker interaction, with a synergy index of 1.66.[20] The probable explanation is that the reported odds ratios were not adjusted for potential confounders.[5]

Studies on mice have indicated toxic and mutagenic effects of areca nut on liver. Liver tumors have been induced by oral administration of areca (betel) nut extract to mice.[21],[22] One of the short comings of the study as noted by the working group was the absence of survival data and indication of duration of the experiment for the treated and control mice.[5],[22] Bhisey et al. fed 54 male and 54 female swiss mice on pan masala. A statistically significant trend in the increase of tumors with dose was observed. The heavy metals, lime, and other unspecified agents might have contributed to the development of tumors in the liver.[23]

On the contrary, did not report any statistically significant increase in tumor incidence in animals treated with areca nut compared with controls.[24] Shirname et al., 1983, too did not report any hepatocellular damage in the mice, all the lessions reported were lung adenocarcinomas. Though the working group in IARC noted that the reason for the difference between the initial number of animals and the number of animals assessed for the presence of tumors is unknown.[5],[25]

Studies on Wistar rats conducted by our department (unpublished data) subjecting them to preparations of various forms of areca nut are in agreement with the studies noted above. The experimental animals showed minimum effects on the liver (most of the changes were reversed over a period of 36 weeks). Most degenerative changes were seen in the pan masala and the raw areca nut group. These changes observed over a period of 36 weeks seem to be self-limiting and reparative. No cases of carcinoma development were seen in either of the two groups.


  Conclusion Top


The results of the study seem to indicate that even long-term chewing of areca nut is not hepatotoxic. Minor alterations in LFTs were well within accepted limits, and no overt biochemical signs of liver damage were detected. There is a need for a more comprehensive study with larger sample size to substantially arrive at a definite correlation of the effect of areca nut chewing on the liver.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Gupta PC, Warnakulasuriya S. Global epidemiology of areca nut usage. Addict Biol 2002;7:77-83.  Back to cited text no. 1
    
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Patidar KA, Parwani R, Wanjari SP, Patidar AP. Various terminologies associated with areca nut and tobacco chewing: A review. J Oral Maxillofac Pathol 2015;19:69-76.  Back to cited text no. 4
[PUBMED]  Medknow Journal  
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IARC Working Group. Betel-quid and areca-nut chewing and some areca-nut-derived nitrosamines: IARC Monographs on the evaluation of carcinogenic risks to humans 2004;85:1182-3.  Back to cited text no. 5
    
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Hu CW, Chao MR. Direct-acting DNA alkylating agents present in aqueous extracts of areca nut and its products. Chem Res Toxicol 2012;25:2386-92.  Back to cited text no. 6
    
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Giannini EG, Testa R, Savarino V. Liver enzyme alteration: A guide for clinicians. CMAJ 2005;172:367-79.  Back to cited text no. 7
    
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Zain RB, Ikeda N, Gupta PC, Warnakulasuriya S, van Wyk CW, Shrestha P, et al. Oral mucosal lesions associated with betel quid, areca nut and tobacco chewing habits: Consensus from a workshop held in Kuala Lumpur, Malaysia, November 25-27, 1996. J Oral Pathol Med 1999;28:1-4.  Back to cited text no. 8
    
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Gao J, Xie L, Yang WS, Zhang W, Gao S, Wang J, et al. Risk factors of hepatocellular carcinoma – Current status and perspectives. Asian Pac J Cancer Prev 2012;13:743-52.  Back to cited text no. 9
    
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Nelson BS, Heischober B. Betel nut: A common drug used by naturalized citizens from India, Far East Asia, and the South Pacific Islands. Ann Emerg Med 1999;34:238-43.  Back to cited text no. 10
    
11.
Chang EE, Miao ZF, Lee WJ, Chao HR, Li LA, Wang YF, et al. Arecoline inhibits the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1A1 activation in human hepatoma cells. J Hazard Mater 2007;146:356-61.  Back to cited text no. 11
    
12.
Nair J, Ohshima H, Friesen M, Croisy A, Bhide SV, Bartsch H. Tobacco-specific and betel nut-specific N-nitroso compounds: Occurrence in saliva and urine of betel quid chewers and formation in vitro by nitrosation of betel quid. Carcinogenesis 1985;6:295-303.  Back to cited text no. 12
    
13.
Sarma AB, Chakrabarti J, Chakrabarti A, Banerjee TS, Roy D, Mukherjee D, et al. Evaluation of pan masala for toxic effects on liver and other organs. Food Chem Toxicol 1992;30:161-3.  Back to cited text no. 13
    
14.
Raisuddin S, Misra JK. Aflatoxin in betel nut and its control by use of food preservatives. Food Addit Contam 1991;8:707-12.  Back to cited text no. 14
    
15.
Shahabuddin S, Rao AR, Mustafa AS. Effect of arecoline on the humoral and cell mediated immune responses in mice. Indian J Exp Biol 1980;18:1493-4.  Back to cited text no. 15
    
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Andrade RG Jr., Dalvi LT, Silva JM Jr., Lopes GK, Alonso A, Hermes-Lima M. The antioxidant effect of tannic acid on the in vitro copper-mediated formation of free radicals. Arch Biochem Biophys 2005;437:1-9.  Back to cited text no. 16
    
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Padma PR, Lalitha VS, Amonkar AJ, Bhide SV. Anticarcinogenic effect of betel leaf extract against tobacco carcinogens. Cancer Lett 1989;45:195-202.  Back to cited text no. 17
    
18.
Padma PR, Amonkar AJ, Bhide SV. Antimutagenic effects of betel leaf extract against the mutagenicity of two tobacco-specific N-nitrosamines. Mutagenesis 1989;4:154-6.  Back to cited text no. 18
    
19.
Sun CA, Wu DM, Lin CC, Lu SN, You SL, Wang LY, et al. Incidence and cofactors of hepatitis C virus-related hepatocellular carcinoma: A prospective study of 12,008 men in Taiwan. Am J Epidemiol 2003;157:674-82.  Back to cited text no. 19
    
20.
Tsai JF, Chuang LY, Jeng JE, Ho MS, Hsieh MY, Lin ZY, et al. Betel quid chewing as a risk factor for hepatocellular carcinoma: A case-control study. Br J Cancer 2001;84:709-13.  Back to cited text no. 20
    
21.
Parkin DM, Srivatanakul P, Khlat M, Chenvidhya D, Chotiwan P, Insiripong S,et al. Liver cancer in Thailand. I. A case-control study of cholangiocarcinoma. Int J Cancer 1991;48:323-8.  Back to cited text no. 21
    
22.
Bhide SV, Shivapurkar NM, Gothoskar SV, Ranadive KJ. Carcinogenicity of betel quid ingredients: Feeding mice with aqueous extract and the polyphenol fraction of betel nut. Br J Cancer 1979;40:922-6.  Back to cited text no. 22
    
23.
Bhisey RA, Ramchandani AG, D'Souza AV, Borges AM, Notani PN. Long-term carcinogenicity of pan masala in Swiss mice. Int J Cancer 1999;83:679-84.  Back to cited text no. 23
    
24.
Pai SR, Shirke AJ, Gothoskar SV. Long-term feeding study in C17 mice administered saccharin coated betel nut and 1,4-dinitrosopiperazine in combination. Carcinogenesis 1981;2:175-7.  Back to cited text no. 24
    
25.
Shirname LP, Menon MM, Nair J, Bhide SV. Correlation of mutagenicity and tumorigenicity of betel quid and its ingredients. Nutr Cancer 1983;5:87-91.  Back to cited text no. 25
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]


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